Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24

Abstract

Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. Many alt-ORFs are co-encoded with canonical proteins in multicistronic configurations, but few of their functions are known. Here, we report the detection of alt-RPL36, a protein co-encoded with human RPL36. Alt-RPL36 partially localizes to the endoplasmic reticulum, where it interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 increases plasma membrane PI(4,5)P2 levels, upregulates PI3K-AKT-mTOR signaling, and increases cell size. Alt-RPL36 contains four phosphoserine residues, point mutations of which abolish interaction with TMEM24 and, consequently, alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as an upstream regulator of PI3K-AKT-mTOR signaling. More broadly, the RPL36 transcript encodes two sequence-independent polypeptides that co-regulate translation via different molecular mechanisms, expanding our knowledge of multicistronic human gene functions.

Nascent alt-protein chemoproteomics reveals a repressor of ribosome biogenesis

“…Western blotting further confirmed the translation of the six alt-proteins (Figure 1g). MINAS-60 and alt-CNPY2 produced two immunoreactive bands, which is due to multiple in-frame start codon for MINAS-60 (vide infra), and may be caused by multiple start sites, phosphorylation or other post-translational modification for alt-CNPY2, analogous to alt-RPL36 8 .…”

“…We then identified the start codon(s) that initiate MINAS-60 translation. Alt-ORFs have been reported to initiate at upstream, non-AUG start codons 8,12,40 and internal, AUG start codons 7,41 . We tested two upstream in-frame non-AUG start codons, A 383 TC and A 386 GG (numbered relative to the first nucleotide of the cDNA), as well as seven internal AUG start codons (numbered ATG1 – ATG7) (Supplementary Figure 3a).…”

“…Cells were harvested and suspended in 1 mL nuclear isolation buffer (10 mM HEPES-KOH pH 7.4, 100 mM KCl, 5 mM MgCl 2 with 0.5% NP40 and Roche Complete protease inhibitor cocktail tablets (Roche, 11873580001)), and incubated on ice for 10 min, followed by centrifugation at 3,000 g, 4°C, 3 min. The nuclear pellets were suspended in 1 mL lysis buffer (Tris-buffered saline (TBS) with 1% Triton X-100 and Roche Complete protease inhibitor cocktail tablets), followed with sonication and immunoprecipitation as previously described 8 . After the final wash, elution was in 40 µL of 3× FLAG peptide (Sigma, F4799), at a final concentration of 100 µg/mL in lysis buffer at 4°C for 1 h. The eluted proteins were subjected to SDS-PAGE separation prior to LC-MS/MS analysis.…”

“…Because bottom-up proteomics did not directly identify the MINAS-60 Nterminus, we experimentally identified the start codons that initiate MINAS-60 translation. Alt-ORFs have been reported to initiate at upstream, non-AUG start codons 8,34,35 and internal, AUG start codons 7,36 , so we tested both possibilities.…”

“…Fewer alt-proteins have been well-defined, including alt-FUS, which cooperates with FUS in formation of cytotoxic aggregates 7 , and alt-RPL36, which regulates the PI3K-AKT-mTOR pathway 8 . Recently, genome-scale CRISPR screens revealed that hundreds of smORFs regulate human cell growth and survival 9,10 .…”